摘要
目的探讨联合应用RNA干扰与干细胞移植技术在肝纤维化治疗中的应用。方法采用全骨髓贴壁培养法分离培养大鼠骨髓间充质干细胞(BMSCs),并进行流式细胞学鉴定。按使用慢病毒包装系统建立沉默基质金属蛋白酶抑制剂-1(TIMP-1)基因不同位点分为三组:BMSCs株SH1组(LV-3-TIMP-1-rat-246),SH2组(LV-3-TIMP-1-rat-142),SH3组(LV-3-TIMP-1-rat-373)。同时设立转染空载体组(LV-3-shNC,D组)。观察各组细胞转染前后形态和荧光的表达;抽提蛋白,应用Western blot技术检测TIMP-1蛋白的表达。结果原代培养大鼠BMSCs呈梭形,传代后漩涡状生长。慢病毒转染BMSCs 7d后,各组均可检测到荧光出现,转染前后细胞形态无改变。在3个实验组中,SH3组TIMP-1蛋白表达最低(P<0.05)。结论应用慢病毒包装系统可以建立沉默TIMP-1基因的BMSCs株,为BMSCs联合RNA干扰技术治疗肝纤维化提供了实验基础。
Objective To study the application of combined use of RNA interference and bone marrow mesenchymal stem cells（BMSCs） transplantation in the treatment of liver fibrosis. Methods BMSCs were isolated from bone marrow of 2-3 weeks old SD rats by whole bone marrow adherence method and identified. Three lentiviral expression vectors carrying different shRNA-TIMP-1 gene were transfected into BMSCs, which included SH1 （LV-3-TIMP-1-rat-246, group Sill）, SH2 （LV-3-TIMP-1-rat-142,group SH2） and SH3 （LV-3-TIMP-1-rat-373,group SH3）. An empty vector transfection（LV-3-shNC） was set up as group 13. The morphology of the cells was observed before and after cell transfectiorL After culturing for 7 days, green fluorescent protein was observed with fluorescence microscope. Protein was extracted and TIMP-1 protein expression was detected by Western blot. Results The primary rat bone marrow mesenchymal stem cells were long spindle-like in sharp and showed a characteristic swirling growth after passage. On the 7th day after lentivirus- transfection, strong fluorescence was detected in each group, but BMSCs morphology was not changed compared to before. Western blot analysis showed that TIMP-1 protein expression in group SHe was the lowest. Conclusion Lentiviral vector carrying shRNA-TIMP-1 gene could make BMSCs stably to knockout TIMP-1 gene, which lays an experimental foundation for the treatment of liver fibrosis with a combination of BMSCs transplantation and RNA interference.
出处
《江苏医药》
CAS
2015年第11期1258-1260,F0003,共4页
Jiangsu Medical Journal
基金
无锡市医院管理中心联合攻关项目(YGZX1202)
关键词
核糖核酸干扰
骨髓间充质干细胞
ARNA interference
Bone marrow mesenchymal stem cells
